Hard shell 384 well pcr plates

A qPCR with primers specific to EcN (Table 1) was used to determine the relative abundance of EcN in fecal samples. All qPCR analyses were performed in triplicate in a reaction volume of 10 μL, using Hard-Shell^® 384-Well PCR plates (Bio-Rad). The reaction mixture contained 2x iQ SybrGreen Supermix (Bio-Rad Laboratories B.V., Lunteren, Netherlands), 200 nM of each primer (Table 1), and 2 μL of the DNA template (1 ng/μL). The amplification program consisted of an initial denaturation at 94°C for 10 min followed by 39 cycles of 94°C for 20 s, 60°C for 30 s, and 72°C for 30 s using a CFX384TM thermocycler (Bio-Rad Laboratories B.V., Lunteren, Netherlands). The fluorescent products were detected at the last step of each cycle. Following amplification, melting temperature analysis of PCR products was performed to determine the specificity of the PCR. The melting curves were obtained by slow heating with 0.5°C/min increments from 60 to 95°C with continuous fluorescence reading. For both EcN-specific and total bacterial primers, standard curves for the qPCR assays were prepared with tenfold serially diluted EcN genomic DNA as a template. The EcN qPCR was performed on a subset of the fecal and digesta samples to validate the EcN-specific ASV in the NGS dataset.t

The qRT-PCR was performed as published previously with minor modifications.^{23 (link)} For RNA isolation, ~50 mg of At seedlings after were harvested with liquid nitrogen in the same light condition. Total RNA was isolated using Qiagen Plant RNeasy mini kit followed by cDNA preparation from 1 μg of RNA of each sample using Bio-Rad iScriptTM Reverse Transcription Super-mix. The quantitative RT-PCR (qRT-PCR) was performed using the CFX384 TouchTM Real-time detection system (Bio-Rad Laboratories), following the manual of iTaqTM universal SYBR green supermix. Gene-specific primers were designed using the Primer Quest tool. All reactions were carried out in Hard-shell 384-well PCR plates (provided by Bio-Rad, Cat #: HSP3805) Table 1. The PCR mix and thermocycler program for the qPCR were similar to those done by Singh, 2022.^{23 (link)} Transcripts level were normalized with ACTIN. Each qRT-PCR reaction was performed in three biological replicates and all data were presented as mean ± SEM.Table 1.

List of primers used in qRT-PCR.

GENES	Forward Primer	Reverse Primer
TIR1	TAATTTGGTACCTGACGGATGG	CACCATCCTCTTCAGCCTTATC
IAA14	CCTTCTAAGCCTCCTGCTAAAG	CTTCGCCGCTCTTCTGATTA
ARF7	CCCTCCAAGTTACTGAGCTTTC	GCTGAGGCAACTGAGACATT
LBD16	TCAAACCGGAGGAGGAGTAT	AGCCTGAAGCTCACCTAAATC
NAC1	GGATCTCATCATCCTCCCAATC	CAGCTTCCCATGTTGTCTCT
AIR3	GGATGACATTCCTGGACCTATC	GACCGGGATTCACAGCTAAA
ACTIN	CGATGAAGCTCAATCCAAACGA	CAGAGTCGAGCACAATACCG